This application is a xc2xa7371 national phase filing of PCT/AU99/00499, having an international filing date of Jun. 18, 1999.
Infertility is a great concern to many couples who wish to conceive. The proportion of couples that are unable to conceive naturally is remarkably high. In the USA it is said that some 10-15% of couples of reproductive age are unable to have children, whereas in the United Kingdom the proportion has been estimated at 14%.
In the last 20 years or so some hope has been held out to infertile couples with the development of in vitro fertilisation (IVF) techniques. These IVF techniques generally take the form of stimulating the female to ovulate, contacting collected ova with sperm in vitro and introducing fertilised ova into the uterus. Multiple variations of this general process also exist. Despite considerable research and technical advances in the IVF field the rate of successful pregnancy following IVF treatment is still quite low and is in the order of 15 to 25% per cycle.
Undertaking an IVF program often causes great anguish, especially where there is no resultant successful pregnancy. It is presently believed that the poor success rate for IVF treatment is due to an extraordinarily high rate of early embryonic loss or implantation failure (Weinberg et al., 1988; Lenton et al., 1988).
The low efficacy of IVF, together with its high cost and the associated psychological trauma from repeated treatment failures make it desirable that improvements are made to the procedure. Current methods of increasing pregnancy rates during IVF treatment include placing multiple embryos (2-5) into the uterine cavity. This is not always successful and also carries with it a higher risk of multiple pregnancy.
In most in vitro fertilisation units embryos are transferred to the uterus 2 days after fertilisation (4-8 cells). One view is that the use of embryos at this early stage may contribute significantly to the low pregnancy outcome of IVF programs and that it is more desirable to use embryos at the blastocyst stage reached at day 5-7 of culture. The advantages suggested include improved synchronisation between embryo and uterus and the ability to select better quality embryos over the longer culture period. Blascocyst transfer may also help reduce the number of multiple births resulting from IVF, through allowing the selection of fewer numbers of highly competent embryos per transfer.
Unfortunately in standard culture media the majority of embryos (about 75%) fail to develop beyond the 4-8 stage. Nevertheless with certain clinical indications implantation of human embryos is performed at the blastocyst stage despite the low proportions of embryos that develop to blastocyst. Some recent studies have used co-culture techniques whereby embryos are co-cultured with feeder cells, for example Vero cells, which technique can more than double blastocyst formation rates (Mxc3xa9nxc3xa9zo et al., 1990; Plachot et al., 1995). There have been a number of studies using these co-culture techniques which have shown increased implantation rates after blastocyst transfer (Mxc3xa9nxc3xa9zo et al., 1992), particularly in women with repeated previous implantation failures (Oliveness et al., 1955; Plachot et al., 1955).
Co-culture is time consuming and expensive and concerns have been expressed about possible transfer of disease from contaminated cultures (Oliveness et al., 1955), in particular there is a concern relating to viral contamination which contamination is considered to be virtually impossible to fully eliminate. A safer and more practical approach is to attempt to produce a culture medium able to sustain embryo development through to the blastocyst stage that is independent of co-culture.
One approach to enhance in vitro embryo development without using co-culture techniques is to attempt to define factors that might be used to enhance embryo development in in vitro culture. A number of attempts have been already made to identify factors that might assist and amongst the promising factors are various stimulatory factors known as cytokines. One such factor, leukemia inhibitory factor (LIF) has already been indicated as being positive in this regard for humans (Dunglison et al 1996) and livestock species, U.S. Pat. No. 5,418,159.
One of the many factors also currently under investigation in both animals and humans relative to conception and embryo development is granulocyte-macrophage colony-stimulating factor (GM-CSF). However to date there has been no definite indication that a medium supplemented with GM-CSF would be sufficient to enhance the in vitro development of embryos to the blastocyst stage in a defined culture medium.
GM-CSF is a 23-29 kD glycoprotein which although secreted in a soluble form in vitro, is one of many cytokines known to be sequestered and immobilised in the ECM (extracellular matrix) in vivo through association with heparan sulphate. GM-CSF was originally characterised as a hemopoietic regulator and determinant of the maturation and behaviour of myeloid leukocytes in peripheral tissues. It is now known that GM-CSF is produced by a diversity of cell types including T-lymphocytes, monocytes, macrophages, fibroblasts, endothelial cells and epithelial cells.
The uterine epithelium has been identified by in situ hybridisation and in in vitro cell isolation studies as a major source of GM-CSF in the mouse uterus (Robertson el al 1992, Robertson et al 1994) and human oviduct and uterus (Zhao and Chegini 1994, Giacomini et al 1995). A role for GM-CSF in reproductive processes was supported by studies perturbing the cytokine environment during early pregnancy in vivo (Tartakovsky and Ben Yair, 1991) and experiments showing impaired fertility in genetically GM-CSF deficient mice (Robertson et al 1999).
Studies of radio-labelled ligand binding show clearly that murine blastocysts bind 125I-GM-CSF specifically, indicating that they express at least the low affinity form of the GM-CSF receptor. This conclusion was supported by RT-PCR analysis, which showed that blastocysts express mRNA for the xcex1-subunit of the GM-CSF receptor complex. A similar situation was found to exist in human embryos. GM-CSF-R was expressed at similar levels through the first four days of murine and human embryo development, from fertilisation to blastocyst stage. However mRNA for the xcex2-subunit of the GM-CSF receptor complex was not detected in embryos of either species by the RT-PCR technique. Together, these data suggest that embryos express GM-CSF receptor from at least as early as fertilisation, but that it may be of the low affinity form. The embryo therefore falls into the same category as endothelial cells and other non-hemopoietic cells which exhibit a biological response to GM-CSF despite expressing only low affinity receptors. Although it seems clear in hemopoietic cells that the xcex1-subunit of the GM-CSF receptor cannot on its own transduce proliferative signal, it is not known whether the xcex1-subunit can in some circumstances initiate responses in cells in the absence of the xcex2-subunit. The recent discovery of unconventional forms of the GM-CSF receptor in the human suggests that this may be possible.
It has also been shown that binding of cognate ligands to the GM-CSF receptor a subunit in isolation may mediate increased glucose transport via a phosphorylation-independent pathway (Ding et al., 1994). Recent experiments by the inventor show that culture with recombinant mouse GM-CSF (mGM-CSF) stimulates increased glucose uptake in murine blastocysts, to an extent achievable with known glucose transport stimulants such as insulin-like growth factor-1, suggesting that this cytokine may stimulate metabolism in murine embryos.
There is some evidence to indicate that GM-CSF also participates in regulation of embryonic growth. Conditioned media rich in mGM-CSF have been found to be effective particularly in promoting blastocyst development, particularly in the attachment of hatched blastocysts to serum attachment factors in plastic culture dishes (Robertson et al., 1991). The media was conditioned by cells from LPS activated mouse lung tissue, and contains a number of other factors which could contribute to the embryotrophic activity.
In further studies by the inventor one cell and eight cell mouse embryos were cultured with or without recombinant mouse GM-CSF (rm GM-CSF) in a defined medium, and again there was a significant increase in the rate at which hatched blastocysts attached to the culture dish. The proportion of embryos developing to eight cell or blastocyst stage was not altered by cytokine. The rate at which embryos developed to blastocysts and hatched from the zona pellucida was also similar, regardless of whether cytokine was present or absent.
In further experiments the survival and/or proliferation of blastomeres within developing mouse blastocysts, particularly inner mass cells, was shown to be enhanced by exposure to native GM-CSF in vivo, or by recombinant GM-CSF in vitro (Robertson et al., 1998).
Several groups have reported both positive and negative effects of GM-CSF on various stages of early embryo development. Hill et al. (1987) have found that GM-CSF at high doses ( greater than 1000 U/ml) inhibited the development of 2-cell embryos into morulae. In two studies, ectoplacental cone trophoblast has been found to proliferate in response to GM-CSF (Armstrong and Chaouat 1989; Lea and Clark 1993), but in the second instance an effect was obtained with native but not recombinant cytokine. Haimovoci et al. (1991) found that 250 U/ml or more of GM-CSF inhibited the attachment of blastocysts to fibronectin-coated culture dishes in the absence of serum. Lea and Clark (1993) have reported that recombinant GM-CSF (at between 10 and 100 U/ml) inhibited the incorporation of 3H-thymidine into outgrowing, implanted blastocysts, in a dose dependent manner. Tartakovsky and Ben-Yair (1991) found that systemic GM-CSF administration markedly enhanced early embryonic development in vivo, but did not note any effect of GM-CSF on embryonic development in vitro. These results are difficult to reconcile. However, the differences are likely to be related to the developmental stages examined, the methods for embryo culture, the strains of mice, and the sources and concentrations of cytokine used. For example, some cytokine preparations may contain potentially embryotoxic contaminants such as endotoxin. In addition, there is emerging evidence that there may be more than one mechanism by which GM-CSF is able to exert its effects in target cells, and it is possible that the glycosylation state of the cytokine (which would also be dependant upon its source) may be important for binding to unconventional receptors.
A study of bovine embryos (de Moraes and Hansen 1997) used recombinant bovine GM-CSF (rbGM-CSF) in attempt to enhance embryo development to blastocyst stage. The rbGM-CSF only had a significant impact on the proportion of embryos developing to blastocyst stage at very high levels of 10 ng/ml, and the numbers of embryos tested were relatively low so the results might be viewed with some concern. Additionally it was found however that the proportion of blastocysts that expanded or hatch dropped significantly with the 10 ng/ml rbGM-CSF and 1 ng/ml rbGM-CSF and thus can be seen an adverse effect on the capacity of the blastocysts to be used subsequently as their development had essentially terminated in vitro.
The present invention results from a finding that recombinant human GM-CSF (rhGM-CSF) is effective at substantially increasing the proportion of early embryos that develop to blastocyst and increasing the proportion of those embryos that continue to expanded blastocyst and then hatched blastocyst stages of development. The net result is that a much greater proportion of embryos can now be grown to blastocyst stage and used for implantation in an IVF program in humans.
This contrasts with the mixed findings in other species, whereby only moderate and inconsistent effects on development to blastocyst stage and beyond were reported.
This finding bas implications in the formulation of media for use in in vitro culturing of embryos to blastocyst stage and in methodologies of growing such embryos and in the manner in which IVF programs are conducted. It is anticipated that this invention will lead to a greater success rate in such IVF programs.
Thus in one broad form of a first aspect the invention could be said to reside in a medium for propagation of early stage embryos to blastocyst stage, said medium containing an effective amount of human GM-CSF to increase the percentage of pre-blastocyst embryos which develop to transfer ready blastocysts.
Transfer ready blastocysts are embryos developed to the stage where a blastoceol cavity is clearly evident and comprises greater than 50% of the volume of the embryo. This stage would in the in vivo situation normally be achieved 4-5 days after fertilisation, soon after the embryo has traversed to fallopian tube and arrives in the uterus.
In one form the medium is a serum deprived medium. The serum deprived medium is desirable in so far as the risk of contamination is drastically reduce The term serum deprived when used in this specification refers to a medium that does not include serum, or any partially defined serum fraction as an additive, but may include a medium that includes serum derived components that have been substantially purified from serum, and may or may not have been modified.
In another form the medium might be a fully defined medium.
Most preferably the human GM-CSF is in purified form, and most preferably purified in from a non-animal and nonhuman source, and might thus be purified from a recombinant micro-organism.
The GM-CSF receptors of embryos appears to be somewhat unique in composition compared to GM-CSF receptors elsewhere and it is therefore likely that the support for embryo growth may not require a fully native GM-CSF. The hGM-CSF may thus be modified or altered in any one of a number of ways and may or may not need to be glycosylated. The hGM-CSF may be truncated, include amino acid deletions and substitutions or may be a recombinant molecule with another growth factor such as perhaps LIF.
Where rhGM-CSF is used it is anticipated that the level of rhGM-CSF in the medium as used will be approximately 1 ng/ml which a physiologically normal level. However, ranges of concentration are also possible and it is anticipated that concentrations ranging from about 0.01 ng/ml to about 5 ng/ml will also give an increase depending on the specific activity of the recombinant or native GM-CSF preparation. It will be understood however that it might be found that concentrations outside of this might also lead to a beneficial effect.
A base medium to which the hGM-CSF is added might be any one known to the person skilled in the art.
The media in which this invention might be used can be any media suitable for use for the in vitro support of embryo development and growth. These media might include but are not limited to HTF medium (Quinn, 1985a), IVF50 (Scandinavian IVF Science), S2 (Scandinavian IVF Science), G1.2 (Scandinavian IVF Science), G2.2 (Scandinavian IVF Science) which references are incorporated herein by references in relation to the media.
In a broad form of a second aspect, the invention could be said to reside in a method of growing early stage human embryos to transfer ready blastocysts, including the step of incubating the embryos in vitro in a culture medium containing an effective amount or human GM-CSF for a time and under conditions to increase the proportion of transfer ready blastocysts.
It is anticipated that the early stage embryos will generally be contacted with GM-CSF at an early stage. The early stage of the embryos may be from immediately after fertilisation, through to several days after fertilisation but preferably before 4 days. Most preferably the contact will be within 2 days of fertilisation. It will be understood that these will be at the 2-16 cell, morula or pre-blastocyst. Generally a 1 day embryo will have 2 cells, 3 day is 16 cell or morula and in 4 to 5 days will develop to a blastocyst. A blastocyst is characterised by a clearly visible blastoceol cavity.
It is anticipated that the in vitro growth will be continued until the blastocysts reach the day 5 to 6 stage, however, in certain embodiments of the invention the culturing of the embryo may be to an earlier, or later stage.
In one form this second aspect of the invention comprises culturing of the embryo in a serum deprived medium including human GM-CSF until blastocyst stage is reached, and then transferring to a second medium including human GM-CSF for further culturing
In a broad form of a third aspect the invention could be said to reside in an IVF program comprising the steps of:
contacting an human egg with a human sperm to form a conceptus
growing the resulting conceptus at least after the 8 cell stage embryo has formed in vitro in a defined culture medium containing an effective amount of human GM-CSF for a time and under conditions to increase the chance of achieving a transfer ready blastocyst
transferring the transfer ready blastocyst into a compatible human uterus
For a better understanding, the invention will now be described with reference to a number of examples.